Interferons are important cytokines characterized by antiviral, antiproliferative, and immunomodulatory activities. These activities form a basis for the clinical benefits that have been observed in a number of diseases, including hepatitis, various cancers and multiple sclerosis. The interferons are divided into the type I and type II classes. Interferon β belongs to the class of type I interferons, which also includes interferons α, τ and ω, whereas interferon γ is the only known member of the distinct type II class.
Human interferon β is a regulatory polypeptide with a molecular weight of 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologics. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of interferon β inducible genes which in turn produces effects which can be classified as antiviral, antiproliferative and immunomodulatory.
The amino acid sequence of human interferon β was reported by Taniguchi, Gene 10:11–15, 1980, and in EP 83069, EP 41313 and U.S. Pat. No. 4,686,191.
Crystal structures have been reported for human and murine interferon β, respectively (Proc. Natl. Acad. Sci. USA 94:11813–11818, 1997. J. Mol. Biol. 253:187–207, 1995). They have been reviewed in Cell Mol. Life Sci. 54:1203–1206, 1998.
Relatively few protein-engineered variants of interferon β have been reported (WO 9525170, WO 9848018, U.S. Pat. Nos. 5,545,723, 4,914,033, EP 260350, U.S. Pat. Nos. 4,588,585, 4,769,233, Stewart et al, DNA Vol 6 no2 1987 pp. 119–128, Runkel et al, 1998, Jour. Biol. Chem. 273, No. 14, pp. 8003–8008).
Expression of interferon β in CHO cells has been reported (U.S. Pat. Nos. 4,966,843, 5,376,567 and 5,795,779).
Redlich et al, Proc. Natl. Acad. Sci., USA, Vol. 88, pp. 4040–4044, 1991 disclose immunoreactivity of antibodies against synthetic peptides corresponding to peptide stretches of recombinant human interferon β with the mutation C17S.
Interferon β molecules with a particular glycosylation pattern and methods for their preparation have been reported (EP 287075 and EP 529300).
Various references disclose modification of polypeptides by polymer conjugation or glycosylation. Polymer modification of native interferon β or a C17S variant thereof has been reported (EP 229108, U.S. Pat. No. 5,382,657, EP 593868, U.S. Pat. No. 4,917,888 and WO 99/55377). U.S. Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue. WO 99/67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein. WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, wherein a cysteine residue has been susbstituted with a non-essential amino acid residue located in a specified region of the polypeptide. Interferon β is mentioned as one example of a polypeptide belonging to the growth hormone superfamily. WO 00/23114 discloses glycosylated and pegylated interferon β. WO 00/23472 discloses interferon β fusion proteins. WO 00/26354 discloses a method of producing a glycosylated polypeptide variant with reduced allergenicity, which as compared to a corresponding parent polypeptide comprises at least one additional glycosylation site. U.S. Pat. No. 5,218,092 discloses modification of granulocyte colony stimulating factor (G-CSF) and other polypeptides so as to introduce at least one additional carbohydrate chain as compared to the native polypeptide. Interferon β is mentioned as one example among many polypeptides which allegedly can be modified according to the technology described in U.S. Pat. No. 5,218,092.
Commercial preparations of interferon β are sold under the names Betaseron® (also termed interferon β1b, which is non-glycosylated, produced using recombinant bacterial cells, has a deletion of the N-terminal methionine residue and the C17S mutation), and Avonex™ and Rebif® (also-termed interferon β1a, which is glycosylated, produced using recombinant mammalian cells) for treatment of patients with multiple sclerosis, and have been shown to be effective in reducing the exacerbation rate. More patients treated with these interferon β agents remain exacerbation-free for prolonged periods of time as compared with placebo-treated patients. Furthermore, the accumulation rate of disability is reduced (Neurol. 51:682–689, 1998).
Comparison of interferon β1a and β1b with respect to structure and function has been presented in Pharmaceut. Res. 15:641–649, 1998.
Interferon β is the first therapeutic intervention shown to delay the progression of multiple sclerosis, a relapsing then progressive inflammatory degenerative disease of the central nervous system.
Its mechanism of action, however, remains largely unclear. It appears that interferon β has inhibitory effects on the proliferation of leukocytes and antigen presentation. Furthermore, interferon β may modulate the profile of cytokine production towards an anti-inflammatory phenotype. Finally, interferon β can reduce T-cell migration by inhibiting the activity of T-cell matrix metalloproteases. These activities are likely to act in concert to account for the mechanism of interferon β in MS (Neurol. 51:682–689, 1998).
In addition, interferon β may be used for the treatment of osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma, Hodgkin's disease, breast carcinoma, melanoma, and viral infections such as papilloma virus, viral hepatitis, herpes genitalis, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegalovirus pneumonia, and rhinovirus.
Various side effects are associated with the use of current preparations of interferon β, including injection site reactions, fever, chills, myalgias, arthralgias, and other flu-like symptoms (Clin. Therapeutics, 19:883–893, 1997).
In addition, 6–40% of patients develop neutralizing antibodies to interferon β (Int. Arch. Allergy Immunol. 118:368–371, 1999). It has been shown that development of interferon β-neutralizing antibodies decreases the biological response to interferon β, and cause a trend towards decreased treatment efficacy (Neurol. 50:1266–1272, 1998). Neutralizing antibodies will likely also impede the therapeutic utility of interferon β in connection with treatment of other diseases (Immunol. Immuther. 39:263–268, 1994).
Given the magnitude of side effects with current interferon β products, their association with frequent injection, the risk of developing neutralizing antibodies impeding the desired therapeutic effect of interferon β, and the potential for obtaining more optimal therapeutic interferon β levels with concomitant enhanced therapeutic effect, there is clearly a need for improved interferon β-like molecules.